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Chapter 14

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CHY 205
Mario Estable

Chapter 14: Glycolysis, Gluconeogenesis & the Pentose Phosphate pathway -Glucose is rich with potential energy making in a n excellent fuel. Glc yields a good amt of energy by complete oxidation to CO2 & H2O which proceeds w/ ∆G’°= -2840kJ/mol. It can be efficiently stored in the polymeric form of starch or glycogen so when energy demands inc, glc can be released from these intracellular storage polymers & used to produce ATP either aerobically or anaerobically (II= icRT; van’t Hoff equan; cells can store large quantities of hexose units while maintaining low cytosolic osmolarity). -Glc is also a versatile biochemical precursor capable of supplying intermediates for biosynthetic rxns. Bacteria can use glc to build the C skeletons for every aa, nucleotide, coenz, FA or other metabolic intermediate they need for growth. In animals & vascular plants, glc has 4 major fates: 1) when there’s excess energy, glc can be stored in polymeric form (starch, glycogen, sucrose) & can also be used for synthesis of complex polysacchs, 2) Short term energy needs are met by oxidation of glc to 3C compound (pyruvate) via glycolysis, 3) Glc can be oxidized via pentose phosphate (phosphogluconate) pathway generating NADPH which is used for detoxification & for biosynthesis of lipids & nucleotides, 4) Structural polysacchs, eg. bacterial cell wall, fungi & plants, are derived from glc. 14. 1 Glycolysis -Glycolysis is a universal central pathway in which 1 molecule of glc is converted to 2 molecules of 3C pyruvate. Some of the free energy released from glc is conserved in the form of ATP & NADH, ie. 2 ATP & 2NADH are generated. The glycolytic breakdown of glc is the sole source of metabolic energy in some mammalian tissues & cell types; some plants are modified to store starch; some aquatic plants derive most of their energy from glycolysis; many anaerobic microorganisms are entirely dependent on glycolysis. -Fermentation is a general term for anaerobic degradation of glc, etc to obtain energy, conserved as ATP. B/c living organisms first arose in an atm w/o O2, glycolysis was an anaerobic process. All intermediates of glycolysis are 3-6C phosphorylated compounds. Glycolysis differ among species only in the details of its regulation & in the metabolic fate of the pyruvate formed. Glycolysis is regulated in coordination w/ other pathways. -Importance of glycolysis: glycolysis is a seq of enzyme-catalyzed rxn by which the 6C glc is converted into 2 molecules of 3C pyruvate in 10 steps which occurs in the cytosol. Pyruvate can be further aerobically oxidized ^ can be used as a precursor in biosynthesis. In the process, some of the oxidation free energy is captured by the synthesis of ATP & NADH. Research of glycolysis played a large role in the development of modern biochem: lead to understanding the role of coenzymes, discovery of the pivotal role of ATP, development of methods for enzyme purification & inspiration for the next generations of biochemists. -In the evolution of life, glycolysis was probably 1 of the earliest energy-yielding pathways. It developed before photosynthesis when the atm was still anaerobic thus the task upon early organisms was to figure out a way to extract energy from glc w/o O2. The soln was to activate glc first by transferring a couple of P to it & then collect energy later from the high energy metabolites of the activated glc. I. Pstparatory phase (Phosphorylation of glc to glyceraldehyder-3-P) 1. 1 priming rxn (Phosphorylation of glucose): glc is activated by its phosphorylation at C-6 to yield glc-6-P w/ ATP as the phosphoryl donor. This rxn, irreversible under intracellular conditions is catalyzed by hexokinase (glucokinase in prokaryotes); kinases are enzymes that catalyze the transfer of gamma phosphoryl group of ATP to an acceptor nucleophile & they are a subclass of transferrases. The acceptor in the case of hexokinase is a hexose, D-glc. Hexokinases req Mg2+ for its activity, ie. MgATP2- complex. Hexokinase is a soluble, cystolic pro. *Isozymes are 2/more enzymes that catalyze the same rxn but are encoded by diff genes, eg. Hxk I-IV. Phosphorylation of glc is an ex of a group transfer rxn, ie. Transfer of phosphoryl group. 2. Phosphohexose isomerization: the enzyme phosphohexose isomerise (phosphoglucose isomerase) catalyzes the reversible isomerization of glc-6-P (aldose) to frc-6-P (ketose). The isomerization is critical as the rearrangement of the carbonyl & hydroxyl groups at C1 & C2 are necessary to prelude to next 2 steps. The phosphorylation that occurs in the nxt rxn req the group at C1 first be converted from a carbonyl to an alcohol & in step 4, cleavage of the bond b/w C3 & C4 req a carbonyl group at C2. This step is an example of the 2 major category of biochemical rxn. 3. 2 priming rxn (Phosphorylation of Frc-6-P to Frc-1,6-bisphosphate): phosphofructokinase-1 (PFK-1) catalyzes the transfer of the gamma phosphoryl group from ATP to frc-6-P to yield frc- 1,6-bisphosphate. PFK-1 is distinguished from PFK-2 which catalyzes the formation of 2,6-biP from frc-6-P in a separate pathway. PFK-1 rxn is irreversible under cellular conditions & the 1 st committed step in the glycolytic pathway, frc-1,6-biP is targeted for glycolysis while glc-6-P & frc- 6-P have other possible fates. Some bacteria & protists & maybe all plants have a PFK that uses PPi, not ATP as the phosphoryl group donor to synthesize frc-1,6-biP. PFK-1 is subject to allosteric regulation; PFK-1 is stimulated w/ low [ATP] & high [ADP & AMP] vs PFK-1 is inhibited w/ high [ATP] & low [ADP & AMP], ie. PFK-1 is negatively regulated by cell [ATP]. In some organisms, frc-2,6-biP is a potent allosteric activator of PFK-1; ribulose-5-P is an intermediate in the pentose P pathway that also activates PFK indirectly. An example of a group transfer rxn. 4. Cleavage of Frc-1,6-biP (lysis step): frc-1,6-biphosphate aldolase, aka aldolase catalyzes the reversible aldol condensation. Frc-1,6-biP is cleaved to yield 2 diff triose phosphates, glyceraldehyde-3-P (aldose) & dihydroxyacetone phosphate (ketose). Although the aldolase rxn has a strongly + ∆G’° in the direction of frc-1,6-biP cleavage, at the lower [ ] of reactants present in cells the ∆G is small & the alsdolase rxn is readily reversible. 5. Interconversion of the Triose phosphates: only 1 of the 2 triose P formed by aldolase, glyceraldehyde-3-P can be directly degraded in the next steps of glycolysis. The other product dihydroxyacetone P is rapidly & reversibly converted to glyceraldehyde-3-P by triose phosphate isomerase. After the triose phosphate isomerase rxn, the C atoms derived from C1, C2 & C3 of the starting glc are chemically indistinguishable from C6, C5 & C4; the 2 halves of glc have both yielded glyceraldehyde-3-P. This rxn completes the preparatory phase of glycolysis. The hexose molecule has been phosphorylthed at C1 & C6 & then cleaved to form 2 glyceraldehyde-3-P. This rxn is an example of the 5 major category of biochemical rxns, interconversion. *Carbonyl group C of glyceraldehyde-3-P is designated as C1. *The isomerization in step 2 is critical for setting up the phosphorylation & C—C bond cleavage rxns in steps 3&4. The 2 molecules of ATP are invested before the cleavage of glc. In sum, in the preparatory phase of glycolysis the energy of ATP is invested, raising the free energy content of the intermediates & the C chains of all the metabolized hexoses are converted to a common product, glyceraldehyde-3-P. II. Payoff phase 6. Oxidation of Glyceraldehyde 3-P to 1,3-bisphosphoglycerate (high ∆G’°): catalyzed by st gluceraldehyde 3-P dehydrogenase . This is the 1 of 2 energy conserving rxns that eventually lead to the formation of ATP. The aldehyde group of glyceraldehyde 3-P is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride w/ a phosphoric acid (acyl [RCO-] phosphate). Acyl phosphate has a high ∆G’° of hydrolysis = -49.3kJ/mol. Acyl phosphate is located at C1 of 1,3-bisphosphoglycerate, the product. The dehydrogenase uses an inorganic phosphate, ie. Not a substrate level phosphorylation which reduces NAD+ to NADH + H+. NADH is later used to make ATP. A reversible rxn. The amt of NAD+ in a cell is <10^-5 M & since it’s low, NADH must be recycled. Glycolysis would stop if NADH formed here were not continuously reoxidized & recycled. NAD is attached to the “Rossman fold” of the enzyme (alpha-beta-alpha-beta motifs) 7. Phosphoryl transfer from 1,3-bisphosphoglycerate to ADP: the enzyme phosphoglycerate kinase transfers high energy phosphoryl group from the carboxyl group of 1,3- bisphosphoglycerate to ADP forming ATP & 3-phosphoglycerate, ie. Substrate level phosphorylation. Reversible rxn. Steps 6&7 constitute an energy-coupling process in which 1,3- bisphosphoglycerate is the common intermediate (formed in step 6 which would be endergonic in isolation) & the acyl phosphate group is transferred to ADP in the 2 rxn (step 7) which is strongly exergonic resulting in: Glyceraldehyde 3-P + ADP + Pi + NAD+  3-phosphoglycerate + ATP + NADH + H+ -Step 7, by consuming the product of step 6, keeps the [ ] of that product relatively low at steady state & thereby keeping the Q for the overall energy coupling small which makes ∆G strongly neg, ie. Step 6&7 are coupled through a common intermediate. The outcome then is that the energy released on oxidation of an aldehyde to a carboxylate group is conserved by the coupled formation of ATP from ADP & Pi. The formation of ATP by phosphoryl group transfer from 1,3-bisphosphoglycerate is called substrate-level phosphorylation which involve soluble enzymes & chemical intermediates. 8. Conversion of 3-phosphoglycerate to 2-phosphoglycerate: phosphoglycerate mutase catalyzes the reversible shift of the phosphoryl group b/w C2 & C3 of glycerate w/ the assistance of Mg2+. 1st the phosphoryl group transfer occurs b/w an active site on a His residue & C2 (OH) of the substrate, ie. 3-phosphoglycerate forming 2,3-BPG. Then the phosphoryl group transfer from C3 of substrate to the 1 active site of His occurs; the 2 active site of His acts as a general acid catalyst producing 2-phosphoglycerate & regenerating the phosphorylated enzyme. 2,3-BPG is required in small quantities to initiate the catalyctic cycle & continuously regenerated by that cycle. 9. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate (PEP): This is the 2 energy nd conserving rxn (step 6 is the 1 one) that generates a compound w/ high ∆Gp; enolase promotes the reversible removal of a molecule of H2O from 2-phosphoglycerate to yield PEP (high ∆G’°= -61.9kJ/mol). 10. Transfer of the phosphoryl group from PEP to ADP: catalyzed by pyruvate kinase which req K+ & Mg2+/Mn2+. In this substrate-level phosphorylation, pyruvate 1 appears in its enol form & then tautomerizes rapidly & non-enzymatically to its keto form which predominates at pH &, driving the rxn. The overall rxn has a large, neg ∆G’° due mostly to the spontaneous converstion of the enol form to keto form of pyruvate. ~half of the energy released by PEP hydrolysis (- 61.9kJ/mol) is conserved in the formation of the phosphoanhydride bond of ATP (-30.5kJ/mol) & the rest (-31.4kJ/mol) constitutes the large driving force pushing the rxn toward ATP synthesis. *Overall Balance sheet: Glucose + 2ATP + 2NAD+ + 4ADP + 2 Pi  2 pyruvate + 2ADP + 2NADH + 2H+ + 4ATP + 2H2O Glucose + 2NAD+ + 2ADP + 2Pi  2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O (under aerobic conditions) -The 2NADH fromed by glycolysis in cytosol are, under aerobic conditions, reoxidized to NAD+ by transfer of their e-s to the ETC, in the mitochondria, which then passes these e-s to O2 providing the energy for synthesis of ATP by respiration linked phosphorylation: 2NADH + 2H+ + O2  2NAD+ + 2H2O -In the overall glycolytic process, The C pathway converts 1 glucose  2 pyruvate. The phosphoryl pathway converts 2ADP + 2Pi to 2ATP. The e- pathway transfers 4 e- (2 :H-) from glyceraldehyde 3-P to 2NAD+ generating 2NADH. -Fates of pyruvate: 1. Pyruvate is oxidized (CO2 lost) to yield acetyl-coA which is then oxidized completely to CO2 by the CAC. The e-s from these oxidations are passed to O2 via ETC to form H2O & the energy form the e- transfer rxns drives the synthesis of ATP in the mitochondria. (From glucosepyruvateacetyl-coA4CO2 + 4 H2O under aerobic conditions) 2. Pyruvate undergoes reduction to lactate via lactic acid fermentation. When vigorously contracting skeletal muscle must function under low O2 conditions (hypoxia), NADH can’t be reoxidized to NAD+ but NAD+ is req as an e- acceptor for the further oxidation of pyruvate, thus pyruvate is reduced to lactate , accepting e-s from NADH & thereby generating NAD+ necessary for glycolysis. Retina & erythrocytes perform this even under aerobic conditions & lactate is also a product of glycolysis under anaerobic conditions in some microorganisms. (From glucose2 lactate via fermentation under anaerobic conditions) 3. Pyruvate catabolism that leads to ethanol. In some plants, invertebrates, protists & some microorganisms, eg. yeast, pyruvate is converted under hypoxic/anaerobic conditions to ethanol & CO2 in what is known as ethanol (alcohol) fermentation. (From glucose2 pyruvate2 ethanol + 2 CO2 via ethanol fermentation in yeast) 4. Pyruvate also serves as a precursor in many anabolic rxns. *The ∆G’° of glycolysis= -85kJ/mol. Under std conditions & nonstd conditions that prevail in a cell, glycolysis is an essentially irreversible process driven to completion by a large net dec in free energy. -Glycolysis under tight regulation: Louis Pasteur discovered that both the rate & total amt of glucose consumption were many times greater under anaerobic than aerobic conditions. The ATP yield of glycolysis under anaerobic conditions (2ATP per glc) is much smaller compared to complete oxidation of glc under aerobic conditions, yielding 30-32 ATP thus 15x more glc needs to be consumed anaerobically as aerobically to yield the same amt of ATP. The flux of glc through the glycolytic pathway is regulated to maintain nearly constant ATP levels. Glycolysis is also regulated by hormones, eg. glucagon, epinephrine & insulin & changes in the expression of genes for several glycolytic enzymes. -Otto Warburg in 1928 observed that tumors carry out glycolysis at a much higher rate than normal tissue even when O2 is available known as the “Warburg effect”. Glc uptake & glycolysis by tumors proceed about 10x faster than noncancerous tissue. Most tumor cells grow under hypoxic conditions b/c initially they lack a capillaries to supply O2. To make the same amt of ATP, tumor cells must take up more glc than normal cells, converting it to pyruvate then lactate as they recycle NADH. It is likely that the 2 early steps in normal celltumor cell transformation are: 1) the change to dependence on glycolysis for ATP production & 2) the development of tolerance to a low pH in the extracellular fluid caused by the release of lactic acid, ie. The more aggressive the tumoir the greater the rate of glycolysis. This inc in glycolysis is achieved in part by inc synthesis of glycolytic enzymes p.m. transporters GLUT-1 & GLUT-3 which carry glc into cells. -The heavier reliance of tumors than normal tissue on glycolysis suggests a possibility for anticancer therapy: inhibitors of glycolysis (ie. Hexokinase) might target & kill tumors by depleting their supply of ATP. By preventing the formation of glc 6-P, these chemotherapeutic agents not only deprive tumor cells of glycolytically produced ATP but also prevent the formation of pentose phosphate via pentose P pathway which also begins w/ glc 6-P. w/o pentose P, a cell can’t synthesize nucleotides essential ot DNA & RNA synthesis & thus can’t grow/divide. The relative rates at which tissues take up glc can be used to pinpoint location of tumors; in PET, individuals are injected w/ a harmless isotopically labelled glc analog that is taken up but not metabolized by tissues. The labelled compound, 2-fluoro-2deoxyglucose (FdG), in which the –OH at C2 is replaced w/ 18^F is taken up via GLUT transporters & a good substrate for hexokinase & therefore accumulates as 6-phospho-Fdg b.c its modified in a way that it can’t be used. *Glc transporters & most glycolytic enzymes are overproduced in tumours. Compounds that inhibit hexokinase, glc-6-P dehydrogenase or transketolase block ATP production by glycolysis thus depriving the cancer cell of energy & killing it. -Glc uptake is def in Type I diabetes: the metabolism of glc in mammals is limited by the rate of glc uptake into cells & the phosphorylation by hexokinase. In skeletal muscle, heart & adipose tissue, glc uptake & metabolism depend on the normal release of insulin by pancreatic B cells in response to elevated blood glc. After a meal containing cho, glc accumulates to abnormally high levels in the blood, aka hyperglycemia; unable to take up glc, muscle & fat tissue use FA of stored triacylglycerols as their prinicipal fuel. In the liver, acetyl-coA derived from this FA breakdown is converted to ketone bodies which are exported & are critical to the brain which uses ketone bodies as an alt when glc is n/a. In untreated Type I diabetes, overproduction of ketone bodies lead to accumulation in blood & lowering blood pH which results in ketoacidosis. Insulin reverses this via GLUT-4 moves into the p.m. of hepatocytes & adipocytes, where glc is taken up into the cells & phosphorylated & blood glc levels fall greatly reducing production of ketone bodies. Summary -All glycolytic enzymes are in cytosol & all 10 intermediates are phosphorylated compounds b/w 3-6C. Glycolysis is tightly regulated in coordination w/ other energy-yielding pathways to assure a steady supply of ATP. 14.2 Feeder Pathways for Glycolysis -The most significant feeder pathways for glycolysis are glycogen & starch (endogenous or obtained in diet) and others are disaccharides maltose, lactose, trehalose & sucrose, monosacchs fructose, mannose & galactose. I. Exogenous dietary glycogen or starch: hydrolyzed to monosachhs. Salivary alpha-amylase in the mouth hydrolyzes internal (alpha14) glycosidic linkages of starch producing oligosacchs. In the stomach, salivary alpha-amylase is inactivated by low pH but a 2 form pancreatic alpha- amylase continues digestion in SI yielding mainly maltose (glc-glc) & maltotriose (glc-glc-glc) & limit dextrins which are fragments of amylopectin containing (alpha 16) branch points. Intestinal brush border enzymes degrade maltoses & dextrins to glc & glc is then absorbed into blood. II. Endogenous storage glycogen or starch stored primarily in the liver & skeletal muscle: are degraded by phosphorolysis which is catalyzed by glycogen/starch phosphorylase. These enzymes attack the (alpha14) glycosidic linkage that joins the last 2 glc residues at a non reducing end generating glc1 –P & a polymer of glc 1 unit shorter. Glycogen phosphorylase acts repetitively until it approaches an (alpha16) branching pt where its action stops. A debranching enzyme removes the branches. Glc 1-P produced by glycogen phosphorylase is converted to glc- 6-P by phosphoglucomutase (function similar to phosphoglycerate mutase which involves a biphosphate intermediate). Glc-6-P can enter glycolysis or the pentose P pathway. *The term mutase is given to enzymes that catalyze the transfer of a func group from 1 position ot another in the same molecules; a subclass of isomerases which are enzymes that interconvert stereoisomers. -Worked example 14-1: phosphorolysis produces glc-1-P which is then converted to glc- 6-P w/o expenditure of ATP needed for the formation of glc-6-P from free glc thus only 1 ATP is consumed per glc monomer in the preparatory phase compared w/ 2 ATP when glycolysis starts w/ free glc. The cell therefore gains 3 ATP per glc monomer ( 4 ATP produced in payoff phase minus 1 ATP used in preparatory phase) III. Disacchs must be hydrolyzed to monosacchs before entering cells. Intestinal disacchs & dextrins are hydrolyzed by enzymes attached to the outer surface of the intestinal epithelial cells: *The monosacchs formed are actively transported into epithelial cells & then passed into the blood to be carried to various tissues where they are phosphorylated & enter glycolysis. Dextrin + nH2O  n D
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